Abstract: Review of the Fraser Fir Story

Sponsored by the College of Agriculture & Life Sciences and the College of Natural Resources, NCSU

Fred P. Hain and Leslie Newton, NC State University

Fraser fir, Abies fraseri (Pursh) Poir., is a unique species that occurs naturally at high elevations in only 3 states, the southern Appalachians of North Carolina, Tennessee and Virginia. Seventy five percent of all naturally occurring Fraser fir is located in the Great Smoky Mountain National Park. The tree is the foundation for the western North Carolina Christmas tree industry, which is one of the largest in the nation. Since the mid 1950’s the tree has been under attack, both in natural stands and in Christmas tree plantations, by the balsam woolly adelgid (BWA), Adelges piceae (Homoptera: Adelgidae). While not considered a significant pest in its native range, the adelgid has caused widespread mortality in the southern Appalachians, killing over 90% of mature fir.

The adelgids infests all above ground portions of the tree, but on Fraser fir the attack is most obvious on the tree bole where a whitewashed appearance may occur. BWA has 4 life stages, egg, crawler, nymph, and adult female. The crawler, or first instar nymph, is the only motile stage. Once she inserts her stylet, she becomes sessile, and slowly begins to excrete the white woolly mass that will cover her body and her eggs. Functional males are not present in North America, and reproduction is parthenogenic. The adelgids feed by inserting their stylets into the ray parenchyma cells of the bark tissue. The stylets are only 1.5 mm long, but the tree’s reaction to the wound runs deep into the xylem tissue. This reaction wood is called “rotholz,” or red wood, that ceases to translocate fluids and causes the eventual death of the tree. Other symptoms of a BWA infestation include premature heartwood, resinosis, gouting, bole stiffness, and loss of apical dominance. The loss of apical dominance is particularly critical to the Christmas tree industry because it frequently occurs very early in the infestation, and renders the tree unsuitable for the Christmas tree market. Classical biological control efforts in the 1950’s and 60’s were unsuccessful in controlling the adelgid. Fortunately, BWA prefers to attack mature trees, and Fraser fir is a prolific seed producer. Consequently, the reproduction has come back strong on many sites, and this second growth Fraser fir is now approaching the susceptible age for BWA. A second wave of tree mortality is likely to take place.

Abstract: Update on host resistance to the balsam woolly adelgid: bioassaying for resistance

Leslie Newton, NC State University

Insect host resistance screening requires an effective technique for artificially infesting the material under observation, so that both host responses to the insect and insect responses to the host can be assessed. Past methodologies have relied primarily upon the application of BWA-infested bark pieces to fir trees of various ages by pinning or tying the infested bark to the stem of the tree. This is an effective method, but it is time consuming, requires repeated applications, and the insects on the bark pieces are sometimes damaged in the process. We have developed a method in which infested bolts of fir are suspended over the material to be infested and the crawlers allowed to simply drop onto the material, mimicking the natural process of infestation. This technique can be utilized in both laboratory and greenhouse trials with minimal time involvement and maximum exposure to BWA crawlers. Additionally, we will present a new and relatively nondestructive technique for host resistance screening throughout the native range of Fraser fir by infesting branch tips cut from living trees. This technique has been successfully employed with both seedlings (5 year old) and mature (20 year old) Fraser fir.

Abstract: A methodological approach to elucidate the effect of balsam woolly adelgid infestation on the structure of wood and bark

This is a part of a multidisciplinary group project conducted at NCSU that focuses on the effect of balsam woolly adelgid (BWA) infestation on the chemical composition of Fraser fir bark. Traditional analytical methods used in the natural product chemistry require a significant amount of time and labor to obtain comprehensive information. Therefore, a methodology allowing rapid and informative analysis of a large number of samples has been developed.

A procedure for sequential extraction of bark with solvents of increasing polarity, using percolation of solvents through ground bark in a small glass column, has been established. This method allows efficient fractionation of extractives of various chemical natures and processing of significant numbers of samples in a short time using small amounts of solvents. Efforts were concentrated on extractives from corky bark, the outer-most layer of bark where the insects attack. After preliminary separation of resin canal extractives, which are mainly composed of resin acids and monoterpenoids, two fractions were selected for detailed chemical profiling: substances extracted with CH2Cl2 (lipophilic extractives) and 90% acetone (polyphenols).

Optimized sample derivatization procedures for combined gas chromatography – mass spectrometry (GC-MS) analysis have been developed for Fraser fir bark, and analyses have been conducted on extracts of bark samples from infested and non-infested trees. The major components of lipophilic extractives were fatty acids, most of them (ca 80%) originally in ester form. Among the fatty acids, oleic and linoleic acids were dominant.

An NMR approach developed for comparative analysis of the polyphenolics fraction allows the characterization of the phenolic compounds without tedious chromatographic separation. This fraction consists of predominantly of flavanoids and condensed tannins with small amounts of phenylpropanoids and lignans. Profiling of the polyphenolic extractives using 1H NMR is rapid and quite informative. The structures of the components of interest have been further verified by comprehensive NMR analysis including correlation 2D NMR techniques (HMQC, HMBC, TOCSY) and quantitative 13C NMR.

The resulting complex datasets are being analyzed using principal components analysis (PCA) and other multi-variate statistical approaches to detect statistically significant differences between infested and non-infested samples. Preliminary PCA results show a relatively good reproducibility of the extraction protocol and GC and NMR analyses. BWA infestation status is correlated with differences in the amounts of individual components, but does not result in appearance of new compounds (or complete disappearance of any compounds). Detailed statistical analysis and identification of the statistically valuable components is in progress.

Jennifer Emerson, Len van Zyl, John Frampton and Ross Whetten, NC State University

Fraser fir is a species native to the southeastern United States and is currently threatened by the balsam woolly adelgid, Adelges piceae (Ratz.) (BWA), both in its natural range and in the extensive Christmas tree industry, which brings in tens of millions of dollars per year in rural areas. Due largely to this exotic insect pest, BWA, old growth Fraser fir has been almost entirely destroyed. Efforts to reduce the impact of the adelgid by the use of pesticides are not feasible in natural stands and are very costly in Christmas tree plantations. Current approaches aimed at reducing the impact of BWA on Fraser fir have shifted towards developing a better understanding regarding the molecular mechanisms involved during host-pest resistance interactions. In recent years, new and powerful molecular techniques, such as microarrays, have been developed to understand the responses of plants to environmental stimuli, developmental stages and interactions among organisms. Microarray analysis permits the simultaneous measurement of gene expression (RNA abundance) of thousands of genes. This technique has revolutionized genomic analysis in many biological systems and could provide a great deal of information on the responses of susceptible and tolerant fir species to BWA feeding. As part of an ongoing NCBC grant, a series of microarray studies are currently being performed in an attempt to identify genes involved in the defense mechanisms or reaction to BWA infestation in Fraser fir. In the first study, field collected phloem tissues from Fraser fir trees with varying infestation levels were collected during two growing seasons. This was done in order to compare gene expression between trees with high levels of BWA infestation to those with low or no infestations, as well as to compare trees with varying levels of bark reaction and apical dominance loss due to the infestation. Results from the first year of collection identified four genes with significant differences in expression for varying amounts of apical dominance loss, as well as four genes with significant differences for varying levels of bark reaction to infestation. These genes are not well characterized, but are associated with plant defense hormone signaling and stress responses based on sequence similarity with loblolly pine genes. These results have prompted the next series of experiments aimed at studying gene expression changes in Fraser fir due to the foliar application of four chemicals involved in plant defense signaling, namely methyl jasmonate, salicylic acid, sodium nitroprusside dihydrate and quercitin dihydrate. These four chemical treatments and a control were applied to Fraser fir seedlings in the greenhouse, with foliage being collected at three time points after each chemical treatment. Experiments are in progress. Also in progress is a study of gene expression changes in three fir species having varying levels of resistance to BWA infestation, which were infested with BWA under laboratory conditions. Results from these various microarray studies will provide us with an in-depth understanding regarding the molecular mechanisms involved during plant-pest interactions that will ultimately assist us in identifying possible biomarkers to screen for resistance to BWA infestation.

Population genetics, the examination of the forces that result in evolutionary changes in populations over time, underpins our understanding of genetic processes in rare and threatened forest tree species. It can help us understand how imperilment affects the evolutionary dynamics in a species, and can inform decisions about where to go to conserve genetic variation and to look for genetic resistance to pests and pathogens. Recent and ongoing molecular marker and enzyme marker studies are illuminating the population genetics of three threatened conifer species of eastern North America: Carolina hemlock (Tsuga caroliniana), eastern hemlock (T. canadensis) and Fraser fir (Abies fraseri).

An amplified fragment length polymorphism (AFLP) molecular marker study of Carolina hemlock indicates that this species has a moderate amount of overall genetic variation and a fairly high amount of genetic differentiation among populations. Additionally, the results suggest that a recent nine-population ex situ seed collection has more than adequately conserved the genetic variation present throughout the range of the species.

An allozyme investigation of genetic variation in 20 eastern hemlock populations in the Southeastern United States suggests that the species has a low level of diversity in the region compared to most other conifers, but greater population differentiation. Populations along the eastern periphery and in the Appalachian interior exhibited greater diversity than those to the west of the mountain chain, demonstrating that the glacial refuge of the species was located east of the southern Appalachian Mountains, the area in which ex situ conservation seed collections should be concentrated to capture the greater genetic variation. An ongoing microsatellite marker study, using primer combinations recently isolated from both eastern and Carolina hemlock, should further elucidate the relationships among eastern hemlock populations.

A separate microsatellite marker analyses of Fraser fir reveals that all of the populations of this fragmented species, found on a handful of peaks in the southern Appalachians, have a significant deficiency of heterozygosity and a high degree of inbreeding relative to other conifers. The results detected a significant but relatively small amount of genetic differentiation among Fraser fir populations, and found that populations further apart were less genetically similar. Mount Rogers, the most isolated population, was the most genetically differentiated. These results guided the first comprehensive collection of cones from all 10 Fraser fir populations and subpopulations in 2007, for gene conservation purposes. A separate study, meanwhile, investigated the relationships among Fraser fir and the closely related balsam fir (Abies balsamea) and intermediate fir (Abies balsamea var. phanerolepis). The results suggest that the three taxa, while genetically distinct, are closely enough related to be considered three varieties of the same species, with Fraser fir apparently representing an adaptive extreme of balsam fir that became isolated during post-Pleistocene warming.

Abstract: Ex situ Gene Conservation of Carolina and Eastern Hemlock

Robert Jetton, Camcore, NC State University

Beginning in 2003, Camcore (an International Tree Conservation & Domestication program at N.C. State University) entered into an agreement with the U.S.D.A. Forest Service to conduct a 3-phase project on the ex situ conservation of hemlocks in the eastern U.S. Phase 1 involves seed collections of Carolina hemlock in the mountains and upper Piedmont regions of the Southeastern U.S. and is ongoing. Seed from 84 open-pollinated families was collected from 13 Carolina hemlock provenances in 2003-2007. The seed has been processed and catalogued for long-term storage. Representative samples have been sent to cooperators in Chile and Brazil who are growing Carolina hemlock seedlings in nursery facilities for eventual out-planting into ex situ conservation banks. Similar conservation banks will be established by the USDA Forest Service in the Ozark Mountains of Missouri and Arkansas using seedlings currently under cultivation at the University of Arkansas. The first conservation plantings will be established in 2008 in Chile with plantings in Brazil and the Ozarks to follow in 2010. Phase 2 started in 2005 and is a 4-year project to collect seed from Eastern hemlock populations in the southeastern U.S. As of December 2007, Camcore has collected seed from 101 open-pollinated families in 18 populations with a final goal of collections from 600 families in 60 populations. Similar to Phase 1, Eastern hemlock seed with be processed and catalogued for long-term storage and the establishment of ex situ conservation banks in Chile, Brazil, and the Ozark Mountains. Phase 3 of the project will sample Eastern hemlock populations in the northeastern and upper Midwestern regions of the U.S. and is tentatively scheduled to begin in 2009.

Abstract: Hemlock Response to HWA Infestation

John Frampton, NC State University

Little research has investigated the response of hemlock (Tsuga spp.) to infestation by the hemlock woolly adelgid, Adelges tsugae (Homoptera: adelgidae). Decline and eventual mortality of infested hemlocks is often attributed to extraction of metabolites by the adelgids. However, this process alone probably does not entirely account for hemlock decline and it is likely that host trees marshal some type of response to infestation. The following hypothesis is being tested: “HWA-infested Eastern (T. canadensis Carriére) and Carolina (T. caroliniana Engelmann) hemlocks die from a host response system that culminates in drought-like mortality due to poor water movement through the xylem”. This hypothesized hemlock response is similar to that of Fraser fir (Abies fraseri [Pursh] Poir.) infested by balsam woolly adelgid (A. piceae Ratz.) expect that the response sites for hemlocks are branch tips rather than the main stem. Circumstantial evidence such as brittle branch tips with some red reaction wood supports this hypothesis. Short- and long-term hydraulic function measurements as well as quantification of changes in xylem structure are being assessed on Eastern and Carolina hemlock trees with different levels of infestation. A better understanding of hemlock response to HWA infestation may lead to enhanced control methods and aid in the development of resistance/tolerance screening techniques.

Abstract: Initial Infestation Rate and Fecundity of the Hemlock Woolly Adelgid (Hemiptera: Adelgidae) on Three Species of North American Hemlock (Tsuga spp.)

Robert Jetton, NC State University

The hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae) is an exotic pest that is causing widespread mortality among hemlocks (Tsuga spp.) in the eastern United States. Tsuga species that occur in Asia and the Pacific Northwest are putatively resistant to adelgid infestation while those in the eastern U.S. are considered generally susceptible. The objectives of this study were to determine if a method for artificially inoculating hemlock seedlings with the adelgid is successful at creating infestations of variable density, and if there are differences in initial adelgid infestation rate and fecundity among three species of North American hemlock: eastern hemlock (T. canadensis Carriére) and Carolina hemlock (T. caroliniana Engelmann), which occur in the east, and western hemlock (T. heterophylla Sargent), which occurs in the west. Seedlings received one of two inoculation treatments in a climate-controlled greenhouse; either low inoculation (50 adeglid egg masses per seedling) or high inoculation (100 adelgid egg masses per seedling). Seedlings of all three species were successfully infested with hemlock woolly adelgid. However, populations of variable density did not develop as a result of initial inoculation density. There was variation among hemlock species with respect to woolly adelgid infestation rate and fecundity. The number of live adelgids feeding on eastern hemlock seedlings was significantly higher than on Carolina and western hemlocks. Adelgids that fed successfully on Carolina hemlock had significantly higher fecundity than conspecifics on either eastern or western hemlock, although this varied depending on inoculation treatment.

Abstract: Forest Service Research on Resistance of Hemlock to HWA

Mike Montgomery and Kurt Gottschalk, USDA Forest Service

Adelges tsugae Annand, the hemlock woolly adelgid (HWA), is a serious pest of hemlocks in the eastern United States. Hemlocks native to western North America and Asia are not damaged by this sucking insect. Recent phylogenetic research (Havill et al. 2006) has determined that HWA is native to western North America and Asia, and that the population in the eastern US is non-native and came from Japan. It is hypothesized that in the areas where HWA is native, a combination of host resistance and natural enemies maintain HWA populations below damaging levels.

For more than a decade, the Forest Service has aggressively supported a program to obtain natural enemies from areas where HWA is endemic for establishment as biological controls for HWA in the eastern U. S. This initial emphasis on establishing biological controls was based on the premise that biological control could be achieved more rapidly than increasing host resistance or tolerance to HWA. Recently, the quest for management options has been more balanced and the availability of special funding in 2005-2007 permitted acceleration of research on biological controls, chemical insecticides, and host resistance. Requests for proposals (RFP) issued these three years funded 11 proposals on host resistance research totaling $624,493.

These extramurally funded proposals cover a wide range of topics, including host chemistry and physiology, feeding behavior, tree breeding, tree ring analysis, and field evaluation of tree resistance, and illustrate the need for a multi-disciplinary approach to development of host resistance. Projects funded were (1) Evaluating phytochemicals (terpenoids) in hemlock species and cultivars in relation to host resistance (Villanova University), (2) Single cell EST sequencing to define molecular responses associated with resistance (FS Southern Research Station), (3) Feeding behavior of HWA in relation to host suitability (North Carolina State Univ.), (4) Field evaluations of non-indigenous and local hemlock species (Cornell Univ.), (5) Evaluating hemlock species for resistance, hardiness, and cultural suitability (Penn. State Univ.), (6) Role of twig morphology in resistance (Oregon State Univ.), (7) Production and evaluation of eastern hemlocks with putative resistance (Univ. of Rhode Island), (8) Evaluation of inter-specific hybrids and development of new hybrids (ARS National Arboretum), (9) Dendrochronological analyses for resistance and changes in soil and foliar nutrients in relation to HWA attack (West Virginia Univ.). Most of these studies are in-progress, but below are brief accounts of some research on HWA which was initiated prior to 2006.

The volatile chemistry of hemlocks has been examined by Anthony Lagalante of Villanova University who developed a sampling method to determine the relative content of volatile terpenoids in the needles of seven hemlock species. For all species, 51 terpenoids were identified, with 2 of these—isobornyl acetate and α-pinene, the most abundant terpenoids—accounting for 90% of the total variance in a principal component analysis on untransformed data. With the exception of T. metensiana, which is in a separate phylogenic group from the other species, the grouping of the hemlocks along both axes of the PCA corresponded to their east-to-west geographic locations. When PCA is run on log-transformed data to minimize the influence of the mean size of the variable, T. caroliniana is grouped with the Asian species and T. canadensis is considerably distant from the other species. This grouping corresponds to the phylogeny of Tsuga species. It is suggested that the high level of isobornyl acetate in the eastern North American hemlocks reflects evolutionary pressure from defoliators in the absence of pressure from sucking insects.

Controlled pollinations by Susan Bentz of the National Arboretum, USDA ARS resulted in authentic hybrids between T. caroliniana and T. chinensis. These hybrids are now of sufficient size in a field nursery that their resistance can be evaluated using artificial inoculations of HWA. Preliminary results indicate that the hybrids have resistance to HWA that is intermediate to that of their parents.

Recent understanding of the genetics of worldwide HWA populations and observations on the biology of these populations has made it clear that it should not be assumed that populations of HWA in the eastern US have the same relationships with hemlock species as populations in other regions. Tsuga chinensis generally has light—but sometimes dense—populations of HWA in China, but is virtually immune to HWA when grown in the eastern US. Similarly, T. heterophylla, in western North America, often has dense HWA, but this species seldom is observed with dense populations of the adelgid when growing in eastern US. Conversely, T. canadensis growing in arboreta in western North America seems to be more resistant than the native, western hemlock growing beside it. Greater abundance of natural enemies of HWA in Asia and Western North American has been suggested as an explanation for both native and exotic hemlocks being less damaged in these areas; however, the different genotypes of HWA in these areas may cause less damage to hosts on which they have coevolved. Although the relative resistance of the hemlock species remains unclear, care should be taken to avoid additional introductions of HWA from Asia or Western North America as these may differ in biology from the genotype now present in the eastern US.

Other researchers are looking for putative heritable resistance among eastern and Carolina hemlocks that have escaped damage from HWA. Another tactic is to identify the role of environment on susceptibility of hemlock to HWA, including silvicultural prescriptions to increase tolerance to HWA.